The present invention relates to topical dosage forms containing human plasma fibronectin for use in promoting wound healing. In particular, the invention relates to the healing of chronic venous ulcers.
Fibronectin is a large glycoprotein containing around So carbohydrate. The characteristic form of plasma fibronectin is a disulfide-bonded dimer of 440,000 daltons, each subunit having a molecular weight of about 220,000 daltons. Normally found in plasma at a concentration of about 300 xcexcg/ml, fibronectin is extracted and purified using a method described by Hynes1. Plasma fibronectin is also known by various other names, including cold-insoluble globulin, anti-gelatin factor, cell attachment protein, cell spreading factor, and opsonic xcex12-surface binding glycoprotein. These names reflect biological activities of fibronectin such as cell recruitment, opsonization of particulate debris, and promotion of wound contraction. Reviews on structure and activities of fibronectin have been published elsewhere2,3.
Wound healing is usually divided into three phases: the inflammatory phase, the proliferative phase, and the remodeling phase. Fibronectin has been reported to be involved in each stage of the wound healing process, particularly by creating a scaffold to which the invading cells can adhere. Initially, many mediators, such as fibronectin and fibrinogen, are released to the wound site. Fibronectin promotes inflammatory cells migration into the wound and debris phagocytosis by the monocytes. Thereafter, angiogenesis and reepithelialization take place. At this stage fibronectin exerts chemotactic activity on endothelial cells, and promotes the migration of epithelial cells and fibroblasts onto the basal membrane. Fibronectin also appears to be an essential component of the remodeling phase where it plays a major role in the organization of collagen fibrils. The fibrillar collagen ultimately forms fibrous bundles that greatly enhance the tissue tensile strength, leading to wound closure.
Topically applied plasma fibronectin has been reported as being useful for increasing the rate of wound healing such as in corneal wounds4,5, and leg ulcers6. However, no one has described a suitable topical carrier for use in treating wounds that can ensure the delivery of an effective amount of fibronectin. A major limiting factor in developing an effective topical dosage form of a drug is not only having an active drug, but also having a formulation that allows the passage of the active drug from the carrier (cream, ointment, gel, etc.) into the site of delivery (which in the case of the present invention is a skin wound). Very active drugs, such as growth factors, may have no therapeutic value if the topical formulation does not allow the drug to move from the semi-solid carrier into the wound. Therefore, it would be highly desirable to develop a formulation which would maximize the contact time of the fibronectin with the wound and also control the release of fibronectin to the wound, thereby leading to high absorption values. The present invention provides such a delivery system in the form of aqueous gels and a cream.
The present invention provides aqueous gel formulations and one cream formulation containing fibronectin and their use for the delivery of an effective wound healing amount of fibronectin to a wound site. The gel formulation comprises a water soluble, pharmaceutically acceptable polymer which is prepared from an effective amount of fibronectin. Examples of such compounds include: vinyl polymers, e.g. polyacrylic acid; polyoxyethylene-polyoxypropylene block copolymers, e.g. poloxamer; and cellulose derivatives, e.g. hydroxyropylcellulose (HPC). The polymer provides viscosity values between 50,000 and 1,000,000 cps at room temperature. The cream formulation is prepared from a commercially available cream base i.e., Schering(copyright) base (Schering Canada Inc., Point-Claire, Quebec), which has viscosity values between 60,000 to 80,000 cps at room temperature.
Many advantages are attributed to these dosage forms. Gel and cream formulations of the present invention release effective amounts of a wound healing promoter. Other advantages of gel formulations include: ability to keep the wound moist (which results from the high water content of the gels), ease of application and removal (by washing) from the wound. They also provide a cool feeling when topically applied which can increase patient comfort.
The slow release system of gel formulations of the present invention provides extended release of fibronectin to the wound site. This property of these formulations permits less frequent application to the wound resulting in less disturbance to the healing process. Such formulations maintain fibronectin delivery for up to 24 hours; but according to kinetic data obtained from permeation studies, a xe2x80x9ctwice a dayxe2x80x9d therapeutic schedule is a preferred embodiment of the present invention.
Formulation of topical dosage forms intended for the incorporation of fibronectin should respect several quality criteria. All components of the preparation including solvent, gelling agent and preservative should be nontoxic for the wound and compatible with the drug. The final product should promote optimal release of the drug to its site of action, be of adequate consistency to enhance contact time of the drug with the wound and be sterile.
The preferred formulations of this invention can be used with other wound healing promoters having a composition similar to fibronectin, such as proteins of similar size (extracellular matrix proteins like thrombospondin, laminin, vitronectin, fibrinogen) or smaller size (such as peptides including growth factors). Human or other mammalian wound healing promoters can be used in formulations for veterinary applications.
The preferred formulations can be correlated with the results of evaluating these formulations using an in vitro diffusion cell system consisting of a rigid receptor containing a deepithelialized skin sample, the deepithelialized side facing upwards into a donor compartment and the dermal side facing downwards into a receptor compartment. The deepithelialized skin sample is prepared by removing a 8 xcexcm section from the epidermal surface of the skin using a dermatome (1/10,000 scissor scale) and the dermal side was carefully cleaned of any adhering subcutaneous tissues and/or blood vessels. The receptor compartment is connected to a circulating buffer circuit, with the buffer temperature maintained at 37xc2x0 C. while the skin surface is at about 32xc2x0 C. Preferred formulations will have an xe2x80x9cAbs valuexe2x80x9d of greater than 7.8, preferably at least 13.40.
A preferred method of preparing the gels of the invention is to concentrate human fibronectin in demineralized water, which contains the polymerization promoter (NaOH) at a pH of about 8.0 to about 11.0. For higher concentrated fibronectin gels (0.5-1.0%), it is preferable to lyophilize the fibronectin. In both cases, the resulting solutions have a pH of about 8.0 to about 11.0. When a carbomer gel is to be prepared, a pH of about 9.0 is preferred. In this manner, it is possible to produce highly concentrated, non-precipitating solutions of fibronectin without using buffers, such as saccharides or stabilizers (e.g., albumin). Concentrated solutions of fibronectin, 2 mg/ml to 10 mg/ml, can be reliably achieved using these methods. The same method may be used to produce highly concentrated aqueous formulations of other wound healing promoters and other generally water-insoluble proteins.
The appropriate fibronectin solution is mixed with a concentrated solution of gelling agent. The two solutions are mixed by multiple exchanges under pressure using devices, such as syringes, which do not vigorously agitate the mixture in order to avoid fibronectin precipitation. The mixing devices are connected by an adaptor device.